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Test Code ACARP Acanthamoeba species Molecular Detection, PCR, Ocular

Useful For

Aids in the diagnosis of amebic keratitis in conjunction with clinical findings

Method Name

Real-Time Polymerase Chain Reaction (PCR)/TaqMan DNA Probe Hybridization

Reporting Name

Acanthamoeba species Detection, PCR

Specimen Type

Varies


Advisory Information


Specimens collected using wooden shafted swabs will be canceled.

 

Fluorescein dye may interfere with testing and should not be used prior to collection. Specimens containing fluorescein dye will be canceled.

 

Although verification experiments did not detect Acanthamoeba species DNA in contact lenses from asymptomatic adults, it is possible that PCR may detect asymptomatic colonization/contamination and, therefore, testing should not be performed on asymptomatic individuals.



Necessary Information


Specimen source is required.



Specimen Required


The preferred specimen for Acanthamoeba PCR from an ocular source is corneal scraping or biopsy.

 

Submit only 1 of the following specimens: 

 

Specimen Type: Tissue: Fresh

Sources: Ocular

Container/Tube: Sterile container

Specimen Volume: 5-10 mm

Collection Instructions: Submit tissue in a sterile container with 1 mL of sterile saline, minimal essential media (MEM), or viral transport media. 

    

Specimen Type: Tissue: Formalin-fixed paraffin-embedded (FFPE)

Sources: Ocular

Container/Tube: Sterile container      

Specimen Volume: 5-10 mm

Collection Instructions: Cut tissue into five, 10-micron sections and place in a sterile container. 

 

Specimen Type: Scrapings/Swabs

Sources: Eye, ocular, cornea

Container/Tube: Sterile container

Specimen Volume: 1 mL

Collection Instructions:

1. Collect corneal scrapings using a scalpel or other sharp device to remove the outer layer of cells from the eye.

2. Swish the collection device in 1 mL of sterile saline, minimal essential media (MEM), or viral transport media.

3. Remove the collection device from the collection container before submitting to the lab.

4. Specimens containing scalpel blades will be canceled.

Additional Information: Swabs are not the preferred specimen for this test and may yield false-negative results.

 

Specimen Type: Contact lenses

Container/Tube: Sterile container

Specimen Volume: Entire collection

Collection Instructions:

1. Place entire contact lens in a sterile container with 1 mL sterile saline, contact lens solution, viral transport media, or minimal essential media (MEM). 

2. Right and Left lenses must be submitted individually using multiple sterile containers or in the original contact lens case. Multiple orders must be created. 

3. Indicate Right or Left in the specimen source.

 

Specimen Type: Contact lens solution

Container/Tube: Sterile container

Specimen Volume: 1 mL solution

 

Specimen Type: Contact lens cases without lenses

Container/Tube: Sterile container

Specimen Volume: 1 mL solution or entire case

Additional Information:

1. Depending on the type of case submitted, it may be necessary to test right and left chambers individually. Multiple orders must be created.

2. Indicate Right or Left in the specimen source.


Specimen Minimum Volume

Tissue: 5 mm biopsy
Scrapings: 0.5 mL
Contact Lens Solution: 1 mL

Specimen Stability Information

Specimen Type Temperature Time
Varies Refrigerated 7 days

Reject Due To

Hemolysis

NA

Lipemia

NA

Icterus

NA

Other

Specimens containing scalpel blades, swabs with wooden handles, or fluorescein dye

Clinical Information

Acanthamoeba are ubiquitous, free-living, microscopic amebae that cause rare, but severe, infections of the eye, skin, lungs, and central nervous system (CNS). They are found worldwide in water and soil and may enter the body through inhalation, contamination of wounds, and contact lens use. As many as 24 species comprising 18 genotypes (T1-T18) have been described, although most human infections are due to genotype T4. Given their widespread distribution in the environment, many people will be exposed to Acanthamoeba during their lifetime, but very few will become sick from this exposure.

 

The most common form of Acanthamoeba infection is amebic keratitis (AK). Infection occurs primarily in contact lens wearers due to contamination of lenses, cleaning solutions, or storage cases. Amebae can also enter the cornea following trauma. AK is a painful, subacute corneal infection associated with extensive scarring and blindness if untreated. Cases generally respond to treatment but relapse is common. Compared to corneal infection, involvement of the CNS is rare and seen primarily in severely immunocompromised individuals such as organ transplant recipients and patients with AIDS. CNS infection may also be caused by a related ameba, Balamuthia mandrillaris.

 

AK is usually clinically suspected based on symptoms and confocal ophthalmologic examination. Confirmation of infection is classically identified by microscopic examination and culture of corneal tissue and contact lenses or equipment using tap water agar plate overlain with bacteria as a food source for the amebae. Unfortunately, it must be held and examined for 7 days for maximum sensitivity. PCR provides a more rapid result with similar sensitivity to culture and is, therefore, the preferred method for confirming the clinical diagnosis of AK.

Reference Values

Negative

Interpretation

A positive result indicates the presence of Acanthamoeba species DNA and is consistent with active or recent infection. While positive results are highly specific indicators of disease, they should be correlated with symptoms, clinical findings, and confocal ophthalmologic examination.

Cautions

While this assay is designed to detect symptomatic infection with Acanthamoeba species, the widespread distribution of these free-living microscopic amebae in the environment may contaminate inanimate objects such as contact lenses and cases. Thus, it should be used for patients with a clinical history and ocular symptoms consistent with amebic keratitis.

 

Inadequate specimen collection or improper storage may invalidate test results.

 

Acanthamoeba species DNA may be detectable for an unknown period of time after adequate treatment.

Supportive Data

The following assay verification data supports the use of this assay for clinical testing.

 

Species Inclusivity:

The Acanthamoeba PCR assay detects the 20 different strains of Acanthamoeba, including the genotypes that cause human disease.

 

Accuracy/Diagnostic Sensitivity and Specificity - Fresh specimens:

Results from this PCR assay detecting the 18S rRNA gene of Acanthamoeba species were compared to culture results on 112 contact/ocular specimens. Of the 12 specimens that were positive by culture, 11 were detected by PCR (sensitivity 92%). PCR also detected an additional 2 positive specimens, which were both from the same patient with a clinical diagnosis of amebic keratitis (AK), while 98 specimens were negative by both culture and PCR (specificity 98%).

 

Accuracy/Diagnostic Sensitivity and Specificity - Formalin-fixed paraffin-embedded (FFPE) specimens:

Twenty-four FFPE archived tissue blocks were tested by the Acanthamoeba species PCR assay and results were compared to histopathologic (light microscopic) diagnosis. Fourteen of the tissues had a morphologic diagnosis of acanthamebic keratitis; of these, 12 were positive by PCR (sensitivity 86%). Ten specimens were negative by both histopathology and PCR (specificity 100%).

 

Supplemental Accuracy Data:

Spiking studies were performed using ocular material in transport media (contact lens fluid, MEM), fresh tissue, and FFPE tissue spiked with Acanthamoeba genomic DNA at an approximate concentration of 50 targets/mcL. All samples were then extracted and tested in a blinded fashion. At 50 targets/mcL, 100% of the ocular material, the fresh, and the FFPE tissue were positive by PCR.

 

Analytical Sensitivity/Limit of Detection (LoD):

-The LoD determined with serial dilution of cultured Acanthamoeba cysts (counted using a hemocytometer) was considered to be 1 cyst per processed sample.

-The LoD established using genomic DNA spiked into contact lens solution/MEM transport media is 1.26 target copies/mcL.

-The LoD established using genomic DNA spiked into fresh tissue matrix is 6.5 target copies/mL.

-The LoD established using genomic DNA spiked into FFPE tissue matrix is 5.7 target copies/mL

 

Analytical Specificity:

No PCR signal was obtained from the extracts of 47 bacterial, viral, parasitic, and fungal isolates from similar organisms or from organisms commonly found in the specimens tested.

 

Precision:

Qualitative inter- and intra-assay precision was 100%. All crossing point values were within 2 cycles of the mean.

 

Reference Range:

The reference range is "Negative" for this assay. PCR and culture performed on 291 contact lenses from asymptomatic individuals failed to detect Acanthamoeba DNA or growth.

 

However, PCR may detect Acanthamoeba species colonization due to the widespread distribution of this free-living ameba in the environment, and PCR testing should only be used for patients with a clinical history and ocular symptoms consistent with AK.

                                                 

Reportable Range:

This is a qualitative assay, and the results are reported as negative or positive for targeted Acanthamoeba species.

Clinical Reference

1. Trabelsi H, Dendana A, Sellami A, et al: Pathogenic free-living amoebae: epidemiology and clinical review. Pathol Biol (paris) 2012;60:399-405

2. Thompson PP, Kowalski RP, Shanks RM, Gordon YJ: Validation of real-time PCR for laboratory diagnosis of Acanthamoeba keratitis. J Clin Microbiol 2008;46:3232-3236

Method Description

The assay is performed on the Roche LightCycler (LC) 480 II instrument, following DNA extraction on the Roche MagNA Pure or the Siemens Tissue Preparation System (TPS). The LC 480 II instrument is an automated instrument that amplifies and monitors the development of target nucleic acid (amplicon) after each cycle of PCR.

 

The DNA target for this PCR assay is a gene encoding the nuclear small subunit ribosomal 18S rRNA.

 

The PCR mix contains a forward and reverse primer specific for Acanthamoeba species template amplification and 1 TaqMan probe (CY5). The CY5 probe contains a fluorophore (5'-end) and a quencher (3'-end) in close proximity; the quencher inhibits the fluorescence signal from the fluorophore while the probe is intact. After the probe anneals to the targeted Acanthamoeba 18S rRNA, it is subsequently degraded by a DNA polymerase with 5'-3' exonuclease activity, resulting in release of the fluorophore and production of detectable fluorescent signal.(Qvarnstrom Y, Visvesvara GS, Sriram R, da Silva AJ: Multiplex real-time PCR assay for simultaneous detection of Acanthamoeba spp, Balamuthia mandrillaris, and Naegleria fowleri. J Clin Microbiol 2006;44:3589-3595)

Day(s) and Time(s) Performed

Monday through Saturday

Analytic Time

2 days

Specimen Retention Time

7 Days

Performing Laboratory

Mayo Medical Laboratories in Rochester

Test Classification

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration.

CPT Code Information

87798

LOINC Code Information

Test ID Test Order Name Order LOINC Value
ACARP Acanthamoeba species Detection, PCR 41429-2

 

Result ID Test Result Name Result LOINC Value
SRCAS Specimen Source 31208-2
38058 Acanthamoeba species PCR 41429-2